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Abstract:

Crystalline forms of 4-[2-(4-methylphenylsulfanyl)-phenyl]piperidine and
salts thereof are provided e.g. for the treatment of neuropathic pain.
##STR00001##

Claims:

1. Compound I, which is 4-[2-(4-methylphenyl-sulfanyl)phenyl]piperidine
##STR00003## and pharmaceutically acceptable salts thereof in a
crystalline form provided said compound is not
4-[2-(4-methylphenylsulfanyl)phenyl]-piperidine hydrochloride addition
salt.

2. The compound according to claim 1, which compound is the HBr addition
salt.

3-4. (canceled)

5. The compound according to claim 1, which compound is the DL-lactic
acid addition salt.

6-7. (canceled)

8. The compound according to claim 1, which compound is the glutaric acid
addition salt (1:1).

9-10. (canceled)

11. The compound according to claim 1, which compound is the malonic acid
addition salt (1:1).

12. The compound according to claim 11, which compound is characterized
by peaks in an XRPD at 10.77.degree., 16.70.degree., 19.93.degree. and
24.01.degree.2.theta., or at 6.08.degree., 10.11.degree., 18.25.degree.
and 20.26.degree.2.theta..

13. The compound according to claim 11, which compound is characterised
by an XRPD as depicted in FIG. 9 or 10.

33. The compound of claim 14, which compound is characterized by peaks in
an XRPD at 11.05.degree., 20.1.degree., 20.60.degree. and
25.00.degree.2.theta., or at 7.80.degree., 13.80.degree., 14.10.degree.
and 19.63.degree.2.theta..

34. The compound according to claim 33, which compound is characterised
by an XRPD as depicted in FIG. 17 or 18.

Description:

[0001] The perception of pain is more complicated than a direct
transmission of signals from an injured part of the body to specific
receptors in the brain, and wherein the pain perceived is proportional to
the injury. Rather, damage to peripheral tissue and injury to nerves may
cause alterations in the central neural structures involved in pain
perception affecting subsequent pain sensitivity. This neuroplasticity
may bring about a central sensitization in response to longer lasting
noxious stimuli, which may manifest itself as e.g. chronic pain, i.e.
that the perception of pain remains even after the noxious stimulus has
stopped, or as hyperalgesia, i.e. an increased response to a stimulus,
which is normally painful. On of the more mysterious and dramatic
examples of this is the "phantom limb syndrome", i.e. the persistence of
pain that existed in a limb prior to its amputation. For a recent review
of central neuroplasticity and pain see Melzack et al in Ann. N.Y. Acad.
Sci., 933, 157-174, 2001.

[0002] Chronic pain, such as neuropathic pain manifests itself differently
than other types of pain, e.g. somatic or visceral pain. The pain is
often described as shooting, burning, pins and needles, numb or stabbing.
Common causes of neuropathic pain include alcoholism, amputation, back,
leg and hip problems, chemotherapy, diabetes, HIV, multiple sclerosis,
spine surgery, and herpes zoster virus infection.

[0003] The central component to chronic pain may explain why chronic pain,
such as e.g. neuropathic pain often responds poorly to classical
analgesics, such as non-steroid anti-inflammatory drugs (NSAIDS) and
opioid analgesics. Tricyclic antidepressants (TCA), typified by
amitryline, have become standard for the treatment of neuropatic pain,
and the effect is believed to be mediated by the combined inhibitory
effect on the serotonin transporter and the norepinephrine transporter
[Gin Ther., 26, 951-979, 2004]. More recently, the so-called dual action
antidepressants having an inhibitory effect on both the serotonin and the
norepinephrine reuptake have been used clinically for the treatment of
neuropatic pain [Human Psychopharm., 19, S21-S25, 2004]. Examples of dual
acting antidepressants are venlafaxine and duloxetine, and this class of
antidepressants is often referred to as SNRI.

[0004] Data on the use of selective serotonine reuptake inhibitors (SSRI)
for neuropathic pain is scarce, but generally suggest a limited effect
[Bas. Gin. Pharmacol., 96, 399-409, 2005]. In fact, it has been
hypothesised that SSRI's are only weakly antinociceptive in and of
themselves but that inhibition of the serotonin transporter augments the
antinociceptive effect of norepinephrine reuptake inhibition. This notion
is supported by a review of 22 animal and five human studies showing that
SNRI's have superior antinociceptive effect compared to norepinephrine
reuptake inhibitors, which again are superior to SSRI [Pain Med. 4,
310-316, 2000].

[0005] Recent data on the 5-HT3 antagonist odansetron implies that
5-HT3 antagonists may have an analgesic effect and thus be useful in
the treatment of neuropathic pain [Anesth. Analg., 97, 1474-1478, 2003].

[0006] The use of tricyclic antidepressants is, however, associated with
known, anticholinergic side effects, such as e.g. drowsiness, anxiety,
restlessness, and cognitive and memory difficulties. Hence, there is a
need in the art to find alternative ways of treating neuropatic pain.

[0007] The international patent application published as WO 2003/029232
discloses e.g. the compound
4-[2-(4-methylphenylsulfanyl)phenyl]piperidine as a free base and the
corresponding HCl salt. The compound is reported to be an inhibitor of
the serotonin transporter and the serotonin receptor 2C (5-HT2C),
and is said to be useful for the treatment of affective disorders, e.g.
depression and anxiety.

SUMMARY OF THE INVENTION

[0008] The present inventors have surprisingly found that in addition to
the already known pharmacological profile,
4-[2-(4-methylphenylsulfanyl)-phenyl]piperidine is a potent inhibitor of
the serotonin reuptake and the norepinephrine reuptake, an antagonist of
the serotonin receptor 3 (5-HT3), an antagonist of the serotonin
receptor 2A (5-HT2A), and an inhibitor or the α1
adrenergic receptor, and the compound may as such be useful in treatment
of e.g. chronic of pain. Accordingly, the invention relates to compound
I, which is 4-[2-(4-methylphenyl-sulfanyl)phenyl]piperidine and
pharmaceutically acceptable salts thereof in a crystalline form provided
said compound is not 4-[2-(4-methylphenyl-sulfanyl)phenyl]-piperidine
hydrochloride addition salt.

[0009] In one embodiment, the invention relates to compound I for use in
therapy.

[0010] In one embodiment, the invention relates to a method of treatment
comprising the administration of a therapeutically effective amount of
compound I to a patient in need thereof.

[0011] In one embodiment, the invention relates to a pharmaceutical
composition comprising compound I.

[0012] In one embodiment, the invention relates to the use of compound I
for the manufacture of a medicament.

FIGURES

[0013]FIG. 1: X-ray diffraction pattern of the HBr addition salt of
compound I

[0035]FIG. 23: Dopamine levels in prefrontal cortex upon administration
of compounds of the present invention.

[0036]FIG. 24: Acetylcholine levels in prefrontal cortex upon
administration of compounds of the present invention.

[0037]FIG. 25A+b: Acetylcholine levels in the prefrontal cortex and
ventral hippocampus upon administration of compounds of the present
invention.

DETAILED DESCRIPTION OF THE INVENTION

[0038] The present invention relates to compound I, which is
4-[2-(4-methylphenylsulfanyl)-phenyl]piperidine and pharmaceutically
acceptable salts thereof in a crystalline form provided said compound is
not the hydrochloride addition salt. The structure of
4-[2-(4-methylphenylsulfanyl)-phenyl]piperidine is

##STR00002##

[0039] The pharmacological profile of the compounds of the present
invention is depicted in the examples, but can be summarised as follows.
The compounds are inhibitors of the serotonin and norepinephrine
reuptake; they inhibit the serotonin receptors 2A, 2C and 3; and they
inhibit the α-1 adrenergic receptor.

[0040] In one embodiment, said pharmaceutically acceptable salts are acid
addition salts of acids that are non-toxic. Said salts include salts made
from organic acids, such as maleic, fumaric, benzoic, ascorbic, succinic,
oxalic, bis-methylenesalicylic, methanesulfonic, ethanedisulfonic,
acetic, propionic, tartaric, salicylic, citric, gluconic, lactic, malic,
malonic, mandelic, cinnamic, citraconic, aspartic, stearic, palmitic,
itaconic, glycolic, p-aminobenzoic, glutamic, benzenesulfonic,
theophylline acetic acids, as well as the 8-halotheophyllines, for
example 8-bromotheophylline. Said salts may also be made from inorganic
salts, such as hydrobromic, sulfuric, sulfamic, phosphoric and nitric
acids. Additional useful salts are listed in the table in example 1d
(table 1).

[0041] In one embodiment, the compound of the present invention, i.e. the
compound of formula I, is the HBr addition salt

[0042] In one embodiment, the compound of the present invention is the
DL-lactic acid addition salt, and in particular the 1:1 salt.

[0043] In one embodiment, the compound of the present invention is the
L-aspartic acid addition salt, and in particular the 1:1 salt.

[0044] In one embodiment, the compound of the present invention is the
glutamic acid addition salt, and in particular the 1:1 salt.

[0045] In one embodiment, the compound of the present invention is the
glutaric acid addition salt, and in particular the 1:1 salt.

[0046] In one embodiment, the compound of the present invention is the
malonic acid addition salt, and in particular the 1:1 salt that is found
to exist in two polymorphic modifications α and β of which the
β form is believed to be the most stable based on a lower
solubility.

[0047] In one embodiment, the compounds of the present invention is in a
purified form. The term "purified form" is intended to indicate that the
compound is essentially free of other compounds or other forms, i.e.
polymorphs of said compound, as the case may be.

[0048] Oral dosage forms, and in particular tablets and capsules, are
often preferred by the patients and the medical practitioner due to the
ease of administration and the consequently better compliance. For
tablets and capsules, it is preferable that the active ingredients are
crystalline.

[0049] Crystals of the present invention may exist as solvates, i.e.
crystals wherein solvent molecules form part of the crystal structure.
The solvate may be formed from water, in which case the solvates are
often referred to as hydrates. Alternatively, the solvates may be formed
from other solvents, such as e.g. ethanol, acetone, or ethyl acetate. The
exact amount of solvate often depends on the conditions. For instance,
hydrates will typically loose water as the temperature is increased or as
the relative humidity is decreased. Compounds, which do not change or
which change only little when conditions, such as e.g. humidity change
are generally regarded as better suited for pharmaceutical formulations.
It is noted that the HBr addition salt does not form hydrates when
precipitated from water whereas compounds such as the succinate, malate
and tatrate acid addition salts do.

[0050] Some compounds are hygroscopic, i.e. they absorb water when exposed
to humidity. Hygroscopicity is generally regarded as an undesired
property for compounds, which are to be presented in a pharmaceutical
formulation, in particular in a dry formulation, such as tablets or
capsules. In one embodiment, the invention provides crystals with low
hygroscopicity.

[0051] For oral dosage forms using crystalline active ingredients it is
also beneficial if said crystals are well-defined. In the present
context, the term "well-defined" in particular means that the
stoichiometry is well-defined, i.e. that the ratio between the ions
forming the salt is the ratio between small integers, such as 1:1, 1:2,
2:1, 1:1:1, etc. In one embodiment, the compounds of the present
invention are well-defined crystals.

[0052] The solubility of an active ingredient is also of significance for
the choice of dosage form as it may have a direct impact on
bio-availability. For oral dosage forms, a higher solubility of the
active ingredient is generally believed to be beneficial as it increases
the bio-availability. Some patients, e.g. elderly patients may have
difficulties swallowing tablets, and oral drop solutions may be a
suitable alternative avoiding the need for swallowing tablets. In order
to limit the volume of an oral drop solution, it is necessary to have a
high concentration of the active ingredient in the solution, which again
requires a high solubility of the compound. As shown in table 3,
DL-lactic acid, L-aspartic acid, glutamic acid, glutaric acid and malonic
acid addition salts have exceptionally high solubility.

[0053] Crystal forms impact the filtration and processing properties of a
compound. Needle formed crystals tend to be more difficult to handle in a
production environment as filtration becomes more difficult and time
consuming. The exact crystal form of a given salt may depend e.g. on the
conditions under which the salt was precipitated. The HBr acid addition
salt of the present invention grows needle-shaped, solvated crystals when
precipitated from ethanol, acetic acid and propanol, but crystals of a
non-hydrated form, which are not needle-shaped, when HBr addition salt is
precipitated from water, providing superior filtration properties.

[0054] Table 3 also depicts the Resulting pH, i.e. the pH in the saturated
solution of the salt. This property is of importance because moisture can
never be completely avoided during storage and the accumulation of
moisture will give rise to a pH decrease in or on a tablet comprising a
low Resulting pH salt, which may decrease shell life. Moreover, a salt
with a low resulting pH may give rise to corrosion of process equipment
if tablets are made by wet granulation. The data in table 3 suggest that
the HBr, HCl and adipic acid addition salts may be superior in this
respect.

[0055] In one embodiment, the compound of the present invention is the HBr
addition salt in a crystalline form, in particular in a purified form. In
a further embodiment, said HBr salt has peaks in an X-ray powder
diffractogram (XRPD) at approximately 6.08°, 14.81°,
19.26° and 25.38°2θ, and in particular said HBr salt
has an XRPD as depicted in FIG. 1.

[0056] In one embodiment, the compound of the present invention is the
DL-lactic acid addition salt (1:1) in a crystalline form, in particular
in a purified form. In a further embodiment, said DL-lactic acid addition
salt has peaks in a XRPD at approximately 5.30°, 8.81°,
9.44° and 17.24°2θ, and in particular said DL lactic
acid addition salt has an XRPD as depicted in FIG. 4.

[0057] In one embodiment, the compound of the present invention is the
L-aspartic acid addition salt (1:1) in a crystalline form, in particular
in a purified form. In a further embodiment, said L-aspartic acid
addition salt is unsolvated and has peaks in a XRPD at approximately
11.05°, 20.16°, 20.60°, 25.00°2θ, and
in particular said L-aspartic salt, when mixed with L-aspartic acid, has
an XRPD as depicted in FIG. 17. In one embodiment, said L-aspartic acid
addition salt is a hydrate, in particular in a purified form. In a
further embodiment, said L-aspartic acid addition salt hydrate has peaks
in a XRPD at approximately 7.80°, 13.80°, 14.10°,
19.63°2θ, and in particular said L-aspartic addition salt
hydrate, when mixed with L-aspartic acid, has an XRPD as depicted in FIG.
18.

[0058] In one embodiment, the compound of the present invention is the
glutamic acid addition salt (1:1) in a crystalline form, in particular in
a purified form. In a further embodiment, said glutamic acid addition
salt has peaks in a XRPD at approximately 7.71°, 14.01°,
19.26°, 22.57°2θ, and in particular said glutamic
acid salt, when mixed with glutamic acid monohydrate, has an XRPD as
depicted in FIG. 19.

[0059] In one embodiment, the compound of the present invention is the
malonic acid addition salt (1:1) in a crystalline form, in particular in
a purified form. In a further embodiment, said malonic acid addition salt
is the α-form and has peaks in a XRPD at approximately
10.77°, 16.70°, 19.93°, 24.01°2θ, or
said malonic acid addition salt is the β-form and has peaks in a
XRPD at approximately 6.08°, 10.11°, 18.25°,
20.26°2θ and in particular said malonic acid addition salt
has an XRPD as depicted in FIG. 9 or 10.

[0060] In one embodiment, the compound of the present invention is the
glutaric acid addition salt (1:1) in a crystalline form, in particular in
a purified form. In a further embodiment, said glutaric acid addition
salt has peaks in a XRPD at approximately 9.39°, 11.70°,
14.05°, and 14.58°2θ, and in particular said glutaric
acid addition salt has an XRPD as depicted in FIG. 8.

[0062] In particular, the compounds of the present invention are useful
for the treatment of mood disorders, such as depression associated with
the above listed chronic pain indications.

[0063] Pain is defined by the International Association for the Study of
Pain (IASP) as "an unpleasant sensory and emotional experience associated
with actual or potential tissue damage, or described in terms of such
damage (IASP Classification of chronic pain, 2nd Edition, IASP Press
(2002), 210). Even though pain is always subjective its causes or
syndromes can be classified. "Neuropathic pain" as a subtype is defined
by the IASP as "pain initiated or caused by a primary lesion or
dysfunction in the nervous system".

[0064] Different subtypes of neuropatic pain are recognised by IASP, and
examples are

[0065] Allodynia which is defined as "a pain due to a
stimulus which does not normally provoke pain".

[0066] Causalgia which is
defined as "a syndrome of sustained burning pain, allodynia and
hyperpathia after a traumatic nerve lesion, often combined with vasomotor
and sudomotor dysfunction and later trophic changes".

[0067]
Hyperesthesia, which is defined as "increased sensitivity to stimulation,
excluding the senses".

[0068] Neuralgia, which is defined as "Pain in the
distribution of a nerve or nerves".

[0069] Neuritis, which is defined as
"Inflammation of a nerve or nerves".

[0070] Neuropathy, which is defined
as "a disturbance of function or pathological change in a nerve: in one
nerve mononeuropathy, in several nerves mononeuropthy multiplex, if
diffuse and bilateral, polyneuropathy". Neuropathy may be associated with
e.g. diabetes in which case it is termed diabetic neuropathy.

[0071]
Hyperalgesia, which is defined as "an increased response to a stimulus
which is normally painful".

[0072] Hyperpathia, which is defined as "a
painful syndrome characterized by an abnormally painful reaction to a
stimulus, especially a repetitive stimulus, as well as an increased
threshold". The stimuli evoking the neuropatic pain may be mechanical or
thermal.

[0073] The unique pharmacological profile of the compounds of the present
inventions make them suitable for the treatment of other diseases, which
are not directly related to chronic pain. 5-HT2C receptors are
located e.g. on dopaminergic neurons where activation exerts a tonic
inhibitory influence on the dopamine release, and 5-HT2C antagonists
will effect an increase in the dopamine level. Data presented in example
2E show that compounds of the present invention do, in deed, bring about
a dose dependent increase in the extra cellular dopamine levels in the
brain. On this background it may be hypothesized that 5-HT2C
antagonists are particular well-suited for the treatment of depression
which is refractory to the treatment with selective serotonin reuptake
inhibitors [Psychopharmacol. Bull., 39, 147-166, 2006]. This hypothesis
finds support in several clinical studies showing a combination of
mirtazipine and SSRI to be superior to SSRI alone for the treatment of
depressed patients with an inadequate clinical response (treatment
resistant depression, TRD, or refractory depression) [Psychother.
Psychosom., 75, 139-153, 2006]. Mirtazapine is also a 5-HT2 and a
5-HT3 antagonist, which indicate that compounds exerting serotonin
reuptake inhibition in combination with 5-HT2 and 5-HT3
antagonism, such as compounds of the present invention, are useful for
the treatment of TRD, i.e. will increase the remission rate for patients
suffering from treatment resistant depression.

[0074] Data presented in example 2F and 2G shows that the compounds of the
present invention bring about an increase in the extracellular level of
acetylcholine in the prefrontal cortex and ventral hippocampus. There is
longstanding clinical evidence that increasing the acetylcholine levels
in the brain is a way to treat Alzheimer's disease and cognitive
impairment in general, cf. the use of acetylcholine esterase inhibitors
in the treatment of Alzheimer's disease. On this background, compounds of
the present invention are believed to be useful in the treatment of
Alzheimer's disease and cognitive impairment, and also mood disorders,
such as depression associated with Alzheimer's disease and cognitive
impairment.

[0075] A segment of depressed patients will respond to treatment with e.g.
SSRI in the sense that they will improve on clinically relevant
depression scales, such as MADRD and HAMD, but where other symptoms, such
as sleep disturbances and cognitive impairment remain. In the present
context, these patients are referred to as partial responders. Due to the
above discussed effects on the acetylcholine levels, the compounds of the
present invention are expected to be useful in the treatment of the
cognitive impairment in addition to the depression. Clinical studies have
shown that the compound prazosin, which is an α-1 adrenergic
receptor antagonist reduces sleep disturbances [Biol. Psychiatry, 61,
928-934, 2007]. Moreover, the 5-HT2A and 5-HT2C antagonism of
the compounds of the present invention is also believed to have a
sedative, sleep-improving effect [Neuropharmacol, 33, 467-471, 1994]
wherefore the compounds of the present invention are useful for the
treatment of partial responders, or rephrased that treatment of depressed
patients with compounds of the present invention will reduce the fraction
of partial responders.

[0076] Attention deficit hyperactivity disorder (ADHD) is one of the most
common neurobehavoioral disorders. ADHD is characterised by the presence
of a triad of social and communicative impairments with restricted,
repetitive or stereotyped behaviours. ADHD usually starts in childhood or
adolescence, but symptoms may continue into adulthood. Atomoxetine is
currently the only nonstimulant approved by FDA for the treatment of ADHD
[Drugs, 64, 205-222, 2004]. Atomoxetine is a norepinephrine reuptake
inhibitor, and this suggests that compounds of the present invention may
be used in the treatment of ADHD. In addition, compounds of the present
invention may have a sedative effect due to the α-1 adrenergic
receptor and 5-HT2 antagonism discussed above, which is beneficial
in the treatment of ADHD.

[0077] Melancholia is a particular subtype of depression often connected
to severe depression; this type of depression is also referred to as
melancholic depression. Melancholia is associated with anxiety, dread of
the future, insomnia, and loss of appetite. Compounds that inhibit both
the serotonin and the norepinephrine reuptake, such as e.g. venlafaxine,
have been shown to be particular effective in the treatment of patients
with severe depression and melancholia [Depres. Anxiety, 12, 50-54,
2000]. As discussed above, compounds exerting 5-HT2C antagonism
increase the dopamine level, wherefore such compounds would be expected
to be effective in the treatment of melancholia [Psychpharm. Bull., 39,
147-166, 2006]. Additionally, the α-1 adrenergic receptor and
5-HT2 antagonism of the compounds of the present invention is
expected to help normalise sleep, wherefore said compounds are useful in
the treatment of melancholia.

[0078] FDA has recently approved sertraline and paroxetine, two SSRI's,
for the treatment of post traumatic stress disorder (PTSD). Moreover,
compounds having 5-HT2A antagonistic activity are useful as they are
expected to be able to contain agitation, insomnia and explosiveness in
PTSD patients [Curr opinion Invest. Drug, 4, 37-41, 2003]. Accordingly,
the compounds of the present invention are expected to be useful in the
treatment of PTSD.

[0079] Hot flushes is a symptom associated with the menopausal transition.
Some women may suffer from this to an extent where it interferes with
sleep or activities in general, and where treatment is necessary. Hormone
replacement therapy with estrogen has been established practice for
decades, however, recently concerns have been voiced on side effects,
such as breast cancer and cardiac events. Clinical trials with SSRI and
SNRI have shown that these compounds have an effect on hot flushes,
albeit less than for estrogen [J. Am. Med. Ass., 295, 2057-2071, 2006].
Treatment of hot flushes with compounds inhibiting serotonin and/or
norepinephrine reuptake, e.g. compounds of the present invention could,
however, be an alternative treatment for women who can not or will not
accept estrogen.

[0080] Sleep apnea or obstructive sleep apnea-hyponea syndrome or
obstructive sleep-disordered breathing is a disorder for which an
effective pharmacotherapy remains to be identified. Several studies in
animals, however, suggest that 5-HT3 antagonists, e.g. compounds of
the present invention may be effective in a therapeutic intervention
[Sleep, 21, 131-136, 1998; Sleep, 8, 871, 878, 2001].

[0081] The 5-HT3 antagonist odansetron has recently been shown
effective in the treatment of craving and alcohol and drug abuse [Drug
Alc. Depend., 84, 256-263, 2006; Pharmacol Therapeut., 111, 855-876,
2006]. This would seem to support the notion that 5-HT3 antagonists,
e.g. compounds of the present invention may be useful in the treatment of
craving, such as alcohol, nicotine or carbohydrate craving; and alcohol
and drug abuse.

[0088] In an embodiment, the compound of the invention is administered in
an amount of about 0.001 to about 100 mg/kg body weight per day.

[0089] A typical oral dosage is in the range of from about 0.001 to about
100 mg/kg body weight per day, preferably from about 0.01 to about 50
mg/kg body weight per day, administered in one or more dosages such as 1
to 3 dosages. The exact dosage will depend upon the frequency and mode of
administration, the sex, age, weight and general condition of the subject
treated, the nature and severity of the condition treated and any
concomitant diseases to be treated and other factors evident to those
skilled in the art.

[0090] A typical oral dosage for adults is in the range of 1-100 mg/day of
a compound of the present invention, such as 1-30 mg/day, or 5-25 mg/day.
This may typically be achieved by the administration of 0.1-50 mg, such
as 1-25 mg, such as 1, 5, 10, 15, 20 or 25 mg of the compound of the
present invention once or twice daily.

[0091] A "therapeutically effective amount" of a compound as used herein
means an amount sufficient to cure, alleviate or partially arrest the
clinical manifestations of a given disease and its complications in a
therapeutic intervention comprising the administration of said compound.
An amount adequate to accomplish this is defined as "therapeutically
effective amount". The term also includes amounts sufficient to cure,
alleviate or partially arrest the clinical manifestations of a given
disease and its complications in a treatment comprising the
administration of said compound. Effective amounts for each purpose will
depend on the severity of the disease or injury as well as the weight and
general state of the subject. It will be understood that determining an
appropriate dosage may be achieved using routine experimentation, by
constructing a matrix of values and testing different points in the
matrix, which is all within the ordinary skills of a trained physician.

[0092] The term "treatment" and "treating" as used herein means the
management and care of a patient for the purpose of combating a
condition, such as a disease or a disorder. The term is intended to
include the full spectrum of treatments for a given condition from which
the patient is suffering, such as administration of the active compound
to alleviate the symptoms or complications, to delay the progression of
the disease, disorder or condition, to alleviate or relief the symptoms
and complications, and/or to cure or eliminate the disease, disorder or
condition as well as to prevent the condition, wherein prevention is to
be understood as the management and care of a patient for the purpose of
combating the disease, condition, or disorder and includes the
administration of the active compounds to prevent the onset of the
symptoms or complications. Nonetheless, prophylactic (preventive) and
therapeutic (curative) treatment are two separate aspect of the
invention. The patient to be treated is preferably a mammal, in
particular a human being.

[0096] In one embodiment, the invention relates to compounds of the
present for use as a medicament for the treatment of chronic pain, such
as neuropathic pain

[0097] The compounds of the present invention may be administered alone as
a pure compound or in combination with pharmaceutically acceptable
carriers or excipients, in either single or multiple doses. The
pharmaceutical compositions according to the invention may be formulated
with pharmaceutically acceptable carriers or diluents as well as any
other known adjuvants and excipients in accordance with conventional
techniques such as those disclosed in Remington: The Science and Practice
of Pharmacy, 19 Edition, Gennaro, Ed., Mack Publishing Co., Easton, Pa.,
1995.

[0098] The pharmaceutical compositions may be specifically formulated for
administration by any suitable route such as the oral, rectal, nasal,
pulmonary, topical (including buccal and sublingual), transdermal,
intracisternal, intraperitoneal, vaginal and parenteral (including
subcutaneous, intramuscular, intrathecal, intravenous and intradermal)
route, the oral route being preferred. It will be appreciated that the
preferred route will depend on the general condition and age of the
subject to be treated, the nature of the condition to be treated and the
active ingredient chosen.

[0099] Pharmaceutical compositions for oral administration include solid
dosage forms such as capsules, tablets, dragees, pills, lozenges, powders
and granules. Where appropriate, they can be prepared with coatings.

[0101] Pharmaceutical compositions for parenteral administration include
sterile aqueous and nonaqueous injectable solutions, dispersions,
suspensions or emulsions as well as sterile powders to be reconstituted
in sterile injectable solutions or dispersions prior to use.

[0103] Conveniently, the compounds of the invention are administered in a
unit dosage form containing said compounds in an amount of about 0.1 to
50 mg, such as 1 mg, 5 mg 10 mg, 15 mg, 20 mg or 25 mg of a compound of
the present invention.

[0104] For parenteral routes such as intravenous, intrathecal,
intramuscular and similar administration, typically doses are in the
order of about half the dose employed for oral administration.

[0105] For parenteral administration, solutions of the compound of the
invention in sterile aqueous solution, aqueous propylene glycol, aqueous
vitamin E or sesame or peanut oil may be employed. Such aqueous solutions
should be suitably buffered if necessary and the liquid diluent first
rendered isotonic with sufficient saline or glucose. The aqueous
solutions are particularly suitable for intravenous, intramuscular,
subcutaneous and intraperitoneal administration. The sterile aqueous
media employed are all readily available by standard techniques known to
those skilled in the art.

[0107] Formulations of the present invention suitable for oral
administration may be presented as discrete units such as capsules or
tablets, each containing a predetermined amount of the active ingredient,
and which may include a suitable excipient. Furthermore, the orally
available formulations may be in the form of a powder or granules, a
solution or suspension in an aqueous or non-aqueous liquid, or an
oil-in-water or water-in-oil liquid emulsion.

[0108] If a solid carrier is used for oral administration, the preparation
may be tablet, e.g. placed in a hard gelatine capsule in powder or pellet
form or in the form of a troche or lozenge. The amount of solid carrier
may vary but will usually be from about 25 mg to about 1 g.

[0109] If a liquid carrier is used, the preparation may be in the form of
a syrup, emulsion, soft gelatine capsule or sterile injectable liquid
such as an aqueous or non-aqueous liquid suspension or solution.

[0110] Tablets may be prepared by mixing the active ingredient with
ordinary adjuvants and/or diluents followed by the compression of the
mixture in a conventional tabletting machine. Examples of adjuvants or
diluents comprise: Corn starch, potato starch, talcum, magnesium
stearate, gelatine, lactose, gums, and the like. Any other adjuvants or
additives usually used for such purposes such as colourings, flavourings,
preservatives etc. may be used provided that they are compatible with the
active ingredients.

[0111] Capsules comprising a compound of the present invention may be
prepared by mixing a powder comprising said compound with
microcrystalline cellulose and magnesium stearate and place said powder
in a hard gelatine capsule. Optionally, said capsule may be coloured by
means of a suitable pigment. Typically, capsules will comprise 0.25-20%
of a compound of the present invention, such as 0.5-1.0%, 3.0-4.0%,
14.0-16.0% of a compound of the present invention. These strengths can be
used to conveniently deliver 1, 5, 10, 15, 20 and 25 mg of a compound of
the present invention in a unit dosage form.

[0112] Solutions for injections may be prepared by dissolving the active
ingredient and possible additives in a part of the solvent for injection,
preferably sterile water, adjusting the solution to the desired volume,
sterilising the solution and filling it in suitable ampoules or vials.
Any suitable additive conventionally used in the art may be added, such
as tonicity agents, preservatives, antioxidants, etc.

[0113] Compound I may be prepared as outlined in WO 2003/029232. Salts of
compound I may by addition of an appropriate acid followed by
precipitation. Precipitation may be brought about by e.g. cooling,
removal of solvent, addition of another solvent or a mixture thereof.

[0114] All references, including publications, patent applications, and
patents, cited herein are hereby incorporated by reference in their
entirety and to the same extent as if each reference were individually
and specifically indicated to be incorporated by reference and were set
forth in its entirety herein (to the maximum extent permitted by law),
regardless of any separately provided incorporation of particular
documents made elsewhere herein.

[0115] The use of the terms "a" and "an" and "the" and similar referents
in the context of describing the invention are to be construed to cover
both the singular and the plural, unless otherwise indicated herein or
clearly contradicted by context. For example, the phrase "the compound"
is to be understood as referring to various "compounds" of the invention
or particular described aspect, unless otherwise indicated.

[0116] Unless otherwise indicated, all exact values provided herein are
representative of corresponding approximate values (e.g., all exact
exemplary values provided with respect to a particular factor or
measurement can be considered to also provide a corresponding approximate
measurement, modified by "about," where appropriate).

[0117] The description herein of any aspect or aspect of the invention
using terms such as "comprising", "having," "including," or "containing"
with reference to an element or elements is intended to provide support
for a similar aspect or aspect of the invention that "consists of",
"consists essentially of", or "substantially comprises" that particular
element or elements, unless otherwise stated or clearly contradicted by
context (e.g., a composition described herein as comprising a particular
element should be understood as also describing a composition consisting
of that element, unless otherwise stated or clearly contradicted by
context).

EXAMPLES

[0118] Analytical Methods

[0119] X-Ray powder diffractograms (XRPD) were measured on a PANalytical
X'Pert PRO X-Ray Diffractometer using CuK.sub.α1 radiation. The
samples were measured in reflection mode in the 2θ-range
5-40° using an X'celerator detector. Elemental composition (CHN)
was measured on an Elementar Vario EL instrument from Elementar. About 4
mg of sample was used for each measurement, and the results are given as
mean values of two measurements.

Example 1a

HBr Salt of Compound I

[0120] To 442 grams of stirred and slightly heated (approx. 45° C.)
4-(2-p-Tolylsulfanyl-phenyl)-piperidine-1-carboxylic acid ethyl ester as
an oil was added 545 ml of 33 wt-% HBr in AcOH (5.7 M, 2.5 eqv.). This
mixing gives a 10° C. exotherm. After final addition the reaction
mixture is heated to 80° C. and left for 18 hours. A sample is
withdrawn and analysed by HPLC and if not completed more 33 wt-% HBr in
AcOH must be added. Otherwise the mixture is cooled to 25° C.
making the product 4-(2-p-Tolylsulfanyl-phenyl)-piperidine hydrobromide
to precipitate. After one hour at 25° C. the thick suspension is
added 800 ml diethylether. Stirring is continued for another hour before
the product is isolated by filtration, washed with 400 ml diethylether
and dried in vacuum at 40° C. overnight. The hydrobromide of
compound I was isolated as white solid.

Example 1b

HBr Salt of Compound I

2-(4-tolylsulfanyl)-phenyl bromide

[0121] In a stirred nitrogen covered reactor N-methyl-pyrrolidone, NMP
(4.5 L) was flushed with nitrogen for 20 minutes. 4-Methylbenzenethiol
(900 g, 7.25 mol) was added and then 1,2-dibromobenzene (1709 g, 7.25
mol). Potassium tert-butoxide (813 g, 7.25 mol) was finally added as the
last reactant. The reaction was exothermic giving a temperature rise of
the reaction mixture to 70° C. The reaction mixture was then
heated to 120° C. for 2-3 hours. The reaction mixture was cooled
to room temperature. Ethyl acetate (4 L) was added and aqueous sodium
chloride solution (15%, 2.5 L). The mixture was stirred for 20 minutes.
The aqueous phase was separated and extracted with another portion of
ethyl acetate (2 L). The aqueous phase was separated and the organic
phases were combined and washed with sodium chloride solution (15%, 2.5
L) The organic phase was separated, dried with sodium sulphate and
evaporated at reduced pressure to a red oil which contains 20-30% NMP.
The oil was diluted to twice the volume with methanol and the mixture was
refluxed. More methanol was added until a clear red solution was
obtained. The solution was cooled slowly to room temperature while
seeded. The product crystallises as off white crystals, they were
isolated by filtration and washed with methanol and dried at 40°
C. in a vacuum oven until constant weight.

Ethyl 4-hydroxy-4-(2-(4-tolylsulfanyl)phenyl)-piperidin-1-carboxylate

[0122] In a stirred reactor under nitrogen cover
2-(4-tolylsulfanyl)-phenyl bromide (600 g, 2.15 mol) was suspended in
heptane (4.5 L). At room temperature 10M BuLi in hexane (235 mL, 2.36
mol) was added over 10 minutes. Only a small exotherm was noticed. The
suspension was stirred for 1 hour at ambient temperature and then cooled
down to -40° C. 1-Carbethoxy-4-piperidone (368 g, 2.15 mol)
dissolved in THF (1.5 L) was added at a rate not faster than the reaction
temperature was kept below -40° C. When the reaction has gone to
completion, it was warmed to 0° C. and 1M HCl (1 L) was added
keeping the temperature below 10° C. The acid aqueous phase was
separated and extracted with ethyl acetate (1 L). The organic phases were
combined and extracted with sodium chloride solution (15%, 1 L). The
organic phase was dried over sodium sulphate and evaporated to a semi
crystalline mass. It was slurried with ethyl ether (250 mL) and filtered
off. Dried in an vacuum oven at 40° C. until constant weight.

Ethyl 4-(2-(4-tolylsulfanyl)phenyl)-piperidin-1-carboxylate

[0123] Trifluoroacetic acid (2.8 kg, 24.9 mol) and triethylsilane (362 g,
3.1 mol) was charged in a reactor with an efficient stirrer. Ethyl
4-hydroxy-4-(2-(4-tolylsulfanyl)phenyl)-piperidin-1-carboxylate (462 g,
1.24 mol) was added via a powder funnel in portions. The reaction was
slightly exothermic. The temperature rose to 50° C. After the
addition was finalised the reaction mixture was warmed to 60° C.
for 18 hours. The reaction mixture was cooled down to room temperature.
Toluene (750 mL) and water (750 mL) was added. The organic phase was
isolated and the aqueous phase was extracted with another portion of
toluene (750 mL). The organic phases were combined and washed with sodium
chloride solution (15%, 500 mL) and dried over sodium sulphate. The
sodium sulphate was filtered off, the filtrate evaporated at reduced
pressure to a red oil which was processed further in the next step.

4-(2-(4-tolylsulfanyl)phenyl)-piperidin hydrobromide

[0124] The crude ethyl
4-(2-(4-tolylsulfanyl)phenyl)-piperidin-1-carboxylate as a red oil from
example 3 was mixed in a stirred reactor with hydrobromic acid in acetic
acid (40%, 545 mL, 3.11 mol). The mixture was heated at 80° C. for
18 hours. The reaction mixture was cooled down to room temperature.
During the cooling the product crystallises out. After 1 hour at room
temperature ethyl ether (800 mL) was added to the reaction mixture, and
the mixture was stirred for another hour. The product was filtered off,
washed with ethyl ether and dried in a vacuum oven at 50° C. until
constant weight.

Example 1c

Recrystallisation of the HBr Salt of Compound I

[0125] A mixture of 10.0 grams of the HBr salt of compound I, e.g.
prepared as above, was heated to reflux in 100 ml H2O. The mixture
became clear and fully dissolved at 80-90° C. To the clear
solution was added 1 gram of charcoal and reflux was continued for 15
minutes before filtered and left to cool spontaneously to room
temperature. During the cooling precipitation of white solid took place
and the suspension was stirred for 1 hour at room temperature. Filtration
and drying in vacuum at 40° C. overnight produced 6.9 grams (69%)
of the HBr acid addition salt of compound I. See FIG. 1 for XRPD.
Elemental analysis: 3.92% N, 59.36% C, 6.16% H (theory: 3.85% N, 59.34%
C, 6.09% H)

Example 1d

Preparation of Stock-Solutions of Free Base

[0126] A mixture of 500 ml ethyl acetate and 200 ml H2O was added 50
grams of the HBr salt of compound I producing a two-phased slurry. To
this slurry was added approximately 25 ml conc. NaOH that caused
formation of a clear two-phased solution (pH was measured to 13-14). The
solution was stirred vigorously for 15 minutes and the organic phase was
separated. The organic phase was washed with 200 ml H2O, dried over
Na2SO4, filtered and evaporated in vacuum at 60° C.
producing the free base in 38 grams yield (99%) as an almost colourless
oil.

[0127] Dissolving 10 grams of the oil and adjusting the volume to 150 ml
using ethyl acetate produced a 0.235 M stock-solution in ethyl acetate
from which aliquots of 1.5 ml (100 mg of the free base) was used.

[0128] Dissolving 10 grams of the oil and adjusting the volume to 100 ml
using 96-vol % EtOH produced a 0.353 M stock-solution in EtOH from which
aliquots of 1.0 ml (100 mg of the free base) was used.

Example 1e

Formation of Salts Using Stock-Solutions of the Free Base

[0129] The given aliquots were placed in test tubes and while stirred the
appropriate amount of acid was added as indicated in Table 1. If the acid
was a liquid it was added neat otherwise it was dissolved in the given
solvent prior to addition. After mixing and precipitation stirring was
continued overnight and the precipitate collected by filtration. Before
drying in vacuum at 30° C. a small reference sample was withdrawn
and dried at room temperature without vacuum. This procedure was included
in order to test for solvates. Some results are presented in Table 1.
XRPD diffractograms are shown in FIGS. 1-22, and selected peak positions
are tabulated in Table 2. Table 3 shows the solubilities of compounds of
the present invention in water together with pH in the resulting
saturated solution. The column "Precipitate" shows whether the
precipitate isolated after the solubility determination is identical to
the compound dissolved, which is indicative of the formation of hydrates.

[0130] Aliquots of test compound and rat cortical synaptosome preparation
were pre-incubated for 10 min/37° C., and then added [3H]NE
or [3H]5-HT (final concentration 10 nM). Non-specific uptake was
determined in the presence of 10 μM talsupram or citalopram and the
total uptake was determined in the presence of buffer. Aliquots were
incubated for 15 minutes at 37° C. After the incubation
[3H]NE or [3H]5-HT taken up by synaptosomes was separated by
filtration through Unifilter GF/C, presoaked in 0.1% PEI for 30 minutes,
using a Tomtec Cell Harvester program. Filters were washed and counted in
a Wallac MicroBeta counter.

[0131] At NET compounds of the present invention display an IC50
value of 23 nM. At SERT compounds of the present invention display an
IC50 value of 8 nM.

Example 2B

5-HT2A Antagonism

[0132] Compounds of the present invention were tested for affinities
towards serotonin receptors and was found to exhibit an antagonistic
profile with affinity at 5-HT2A receptors (Ki 54 nM). The
affinity is calculated from Y=100/(1+10.sup.(X-logIC50.sup.)) where Y
denotes % binding and X denotes the concentration of compound. 5
concentrations of compound (1, 10, 30, 100, 1000 nM) were used to
calculate the IC50 value. Ki was calculated from the Cheng Prusoff
equation Ki=(IC50/(1+([L]/Kd)) Affitiny was determined at MDL
Pharmaservices catalogue number 271650.

[0133] In mammalian cells expressing human 5-HT2A receptors compounds
of the present invention display competitive antagonistic properties. The
compounds bind to 5-HT2A receptors with a Ki of <100 nM and in a
functional assay the compounds antagonise 5-HT evoked release of
Ca2+ from intracellular stores with a Kb of 67 nM. A schild analysis
revealed competitive antagonism with a Kb of 100 nM.

[0134] The experiment was carried out as follows. 2 or 3 days before the
experiment CHO cells expressing 250 fmol/mg human 5-HT2A receptors
are plated at a density sufficient to yield a mono-confluent layer on the
day of the experiment. The cells are dye loaded (Ca2+-kit from
Molecular Devices) for 60 minutes at 37° C. in a 5% CO2
incubator at 95% humidity. Basal fluorescence was monitored in a
fluorometric imaging plate reader or FLIPR384 from Molecular Devices
(Sunnyvale, Calif.) with an excitation wavelength of 488 nm and an
emission range of 500 to 560 nm. Lacer intensity was set to a suitable
level to obtain basal values of approximately 8000-10000 fluorescence
units. The variation in basal fluorescence should be less than 10%.
EC50 values are assessed using increasing concentrations of test
compound covering at least 3 decades. pA2 values are assessed challenging
full dose response curves of 5-HT with four different concentrations of
compound (150, 400 1500 and 4000 nM). Kb values were also assessed
challenging 2 decades of concentrations of test substances with EC85
of 5-HT. Test substances are added to the cells 5 minutes before the
5-HT. Ki values are calculated using Cheng-Prusoff equation.

[0136] Compounds of the present invention were tested for affinities
towards the α1A receptor and was found to exhibit an
antagonistic profile with medium affinity for α1A receptors
(Ki=34 nM).

[0137] On the day of the experiments membranes (see below for description
of membrane preparation) are thawed and homogenized in buffer using an
ultra turrax and diluted to the desired concentration (5
μg/well˜5 μg/900 μl, store on ice until use).

[0138] The experiment is initiated by mixing of 50 μl test compound, 50
μl [3H]-Prazosin and 900 μl membranes, and the mixture is
incubated for 20 minutes at 25° C. Non-specific binding is
determined in the presence of 10 μM WB-4101 and the total binding is
determined in the presence of buffer. After the incubation, bound ligand
is separated from unbound by filtration through Unifilter GF/B, presoaked
in 0.1% PEI for 30 minutes, using a Tomtec Cell Harvester program
(D4.2.4). 96 well. Filters are washed 3 times with 1 ml ice-cold buffer,
dried at 50° C. and 35 μl scintillation liquid/well is added to
the filters. Bound radioactivity is counted in a Wallac OY 1450
MicroBeta. The affinity is calculated from
Y=100/(1+10.sup.(X-logIC50.sup.)) where Y denotes % binding and X denotes
the concentration of compound. Concentrations of compound covering 2
decades were used to calculate the IC50 value. Ki was calculated from the
Cheng Prusoff equation Ki=(IC50/(1+([L]/Kd))

[0139] In a functional assay compounds of the present invention
antagonises adrenaline evoked release of Ca2+ from intracellular
stores and a functional assay revealed that compounds were antagonists.

[0140] These experiments were carried out essentially as described below.

[0142] Twenty-four hours prior to assays, CHO cells expressing the human
alpha1A-7 receptors were seeded into 384-well black wall microtiter
plates coated with poly-D-lysine. Culture medium was aspirated and cells
were dye-loaded with 1.5 μM Fluo-4 in assay buffer composed of Hank's
Balanced Salt Solution (138 mM NaCl, 5 mM KCl, 1.3 mM CaCl2, 0.5 mM
MgCl2, 0.4 mM MgSO4, 0.3 mM KH2PO4, 0.3 mM
Na2HPO4, 5.6 mM glucose) plus 20 mM HEPES pH 7.4, 0.05% BSA and
2.5 mM probenicid (50 μl/well) for 1 hour in 5% CO2 at 37°
C. After excess dye was discarded, cells were washed in assay buffer and
layered with a final volume equal to 45 μl/well (or 30 ul/well for
antagonist assay). In the case of antagonist evaluation, antagonist or
vehicle was added at this point as a 15 μl aliquot in 4%
DMSO-containing buffer at 4× the final concentration (final
DMSO=1%), followed by a 20 min incubation. Basal fluorescence was
monitored in a fluorometric imaging plate reader or FLIPR® from
Molecular Devices (Sunnyvale, Calif.) with an excitation wavelength of
488 nm and an emission range of 500 to 560 nm. Laser excitation energy
was adjusted so that basal fluorescence readings were approximately 8,000
relative fluorescent units (RFU). Cells were then stimulated at room
temperature with agonists diluted in assay buffer (15 μl), and RFU
were measured at 1.5 second intervals over a period of 2.5 min. Maximum
change in fluorescence was calculated for each well.
Concentration-response curves derived from the maximum change in
fluorescence were analyzed by nonlinear regression (Hill equation). For
antagonistic determinations, after 20 min of compound incubation (as
above), fixed concentrations of standard agonist serotonin were added.

Example 2E

Increase in Dopamine

[0143] A single injection of compounds of the present invention
dose-dependently increased extracellular DA levels in the rat frontal
cortex. The compound of the present invention at 8.9 mg/kg and 18 mg/kg
s.c., enhanced the DA levels by approximately 100% and 150%,
respectively, above baseline levels as depicted in FIG. 23. Amounts are
calculated as the free base.

[0144] Method.

[0145] Male Sprague-Dawley rats, initially weighing 275-300 g, were used.
The animals were housed under a 12-hr light/dark cycle under controlled
conditions for regular in-door temperature (21±2° C.) and
humidity (55±5%) with food and tap water available ad libitum. For the
three-day treatment experiments osmotic minipumps (Alzet, 2ML1) were
used. The pumps were filled under aseptic conditions and implanted
subcutaneously under sevoflurance anaesthesia. The experiments were
carried out with the minipumps on board. Blood samples for measuring
plasma levels of the test compound after 3 days of treatment were
collected at the end of the experiments.

[0146] Surgery and Microdialysis Experiments.

[0147] Animals were anaesthetised with hypnorm/dormicum (2 ml/kg) and
intracerebral guide cannulas (CMA/12) were stereotaxically implanted into
the hippocampus, positioning the dialysis probe tip in the ventral
hippocampus (co-ordinates: 5.6 mm anterior to bregma, lateral -5.0 mm,
7.0 mm ventral to dura or in the frontal cortex (co-ordinates: 3.2 mm
anterior to bregma; lateral, 3.0 mm; 4.0 mm ventral to dura). Anchor
screws and acrylic cement were sued for fixation of the guide cannulas.
The body temperature of the animals was monitored by rectal probe and
maintained at 37° C. The rats were allowed to recover from surgery
for 2 days, housed singly in cages. On the day of the experiment a
microdialysis probe (CMA/12, 0.5 mm diameter, 3 mm length) was inserted
through the guide cannula. The probes were connected via a dual channel
swivel to a microinjection pump. Perfusion of the microdialysis probe
with filtered Ringer solution (145 mm NaCl, 3 mM KCl, 1 mM MgCl2,
1,2 mM CaCl2) was begun shortly before insertion of the probe into
the brain and continued for the duration of the experiment at a constant
flow rate of 1 (1,3) μL/min. After 180 min of stabilisation, the
experiments were initiated. Dialysates were collected every 20 (30) min.

[0148] After the experiments the rats were sacrificed by decapitation,
their brains removed, frozen and sliced for probe placement verification.

[0151] The experiment was designed to evaluate the effects of compounds of
the present invention on extracellular levels of acetylcholine in the
prefrontal cortex of freely-moving rats.

[0152] Male Wistar rats (280-350 g; Harlan, Zeist, The Netherlands) were
used for the experiments. Rats were individually housed in plastic cages
(30×30×40 cm) and had ad libitum access to food and water.

[0153] Rats were anesthetized using isoflurane (2%, 400 mL/min N2O,
400 ml/min O2). Lidocain (10% m/v) was used for local anesthesia.
Each animal was placed into a stereotaxic frame (Kopf instruments, USA),
and home-made I-shaped probes (Hospal AN 69 membrane, 4 mm exposed
surface) were inserted into the medial prefrontal cortex (mPFC) using the
rat brain atlas of Paxinos and Watson (1982). Coordinates for the tip of
the probe was mPFC [AP=3.4 mm, L=-0.8 mm, V=5.0 mm]. The probe was then
fixed to the skull with dental cement and a srew. Flunixin (1 mg/kg s.c.)
was administered as post-operative analgesic.

[0154] Experiments were carried out 24-48 hours after surgery. On the day
of the experiment, rats were connected with flexible PEEK tubing to
microperfusion pumps (CMA 102), and the dialysis probes were perfused
with a Ringer buffer containing 147 mM NaCl, 3.0 mM KCl, 1.2 mM
CaCl2, and 1.2 mM MgCl2, at a flow rate of 1.5 μL/min.
Microdialysis samples were collected at 30 min intervals into mini-vials
containing 55 μL 0.02 M formic acid for determination of
acetylcholine. Samples were collected by an automated fraction collector
(CMA 142), and stored at -80° C. until analyzed. After completion
of the experiments the rats were sacrificed. The brains were removed and
cured in paraformaldehyde solution (4% m/v). The positioning of each
probe was verified histologically according to Paxinos and Watson (1982),
by making coronal sections of the brain.

[0155] The test compound was dissolved in 10%
2-OH-propyl-beta-cyclodextrin and administration occurred by subcutaneous
injections of 5 mL/kg volumes in different doses.

[0157] Aliquots (25 μL) were injected onto the HPLC column by an
automated sample injector (PerkinElmer Instruments, series 200).
Chromatographic separation was performed on a reverse-phase
150×2.00 mm (4 μm) analytical column (Phenomenex Synergy MAX-RP,
Bester) protected by a 4×2.0 mm guard column (Phenomenex Synergy
MAX-RP AJO-6073, Bester), both held at a temperature of 30° C. The
mobile phase (isocratic) consisted of ultrapurified water (UP),
acetonitrile (ACN), and trifluoroacetic acid (TFA)
(UP:ACN:TFA=95.0:0.5:0.1 v/v/v %). Mobile phase was run through the
system at a flow rate of 0.300 mL/min by an HPLC pump (PerkinElmer
Instruments, series 200 micro pump).

[0158] The LC/MS analyses were performed using a API 4000 MS/MS system
consisting of a API 4000 MS/MS detector and a Turbo Ion Spray interface
(both from Applied Biosystems, the Netherlands). The acquisitions were
performed in positive ionization mode, with ion spray voltage set at 5.5
kV, the nebulizer gas pressure at 50 psig (on a SCIEX scale 0-90) with a
probe temperature of 600° C. The instrument was operated in
multiple-reaction-monitoring (MRM) mode for detection of acetylcholine
(precursor 146.1 Da, product 86.8 Da). The collision energy was 21.0 eV,
and the collision gas (nitrogen) pressure was held at 7 (on a SCIEX scale
of 0-12). Data were calibrated and quantitated using the Analyst® data
system (Applied Biosystem, version 1.2).

[0159] Two consecutive microdialysis samples with less then 50% variation
were taken as baseline levels and set at 100%. Changes in acetylcholine
concentration were expressed as percent of baseline within the same
subject. The data are shown in FIG. 24

Example 2G

Increase in Acetylcholine

[0160] The experiment was designed to evaluate the effects of compounds of
the present invention on extracellular levels of acetylcholine in the
prefrontal cortex and ventral hippocampus of freely-moving rats.

[0161] Male Sprague-Dawley rats, initially weighing 275-300 g, were used.
The animals were housed under a 12-hr light/dark cycle under controlled
conditions for regular in-door temperature (21±2° C.) and
humidity (55±5%) with food and tap water available ad libitum.

[0162] Surgery and Microdialysis Experiments

[0163] Rats were anaesthetised with hypnorm/dormicum (2 ml/kg) and
intracerebral guide cannulas (CMA/12) were stereotaxically implanted into
the hippocampus, aiming to position the dialysis probe tip in the ventral
hippocampus (co-ordinates: 5.6 mm posterior to bregma, lateral -5.0 mm,
7.0 mm ventral to dura or in the frontal cortex (co-ordinates: 3.2 mm
anterior to bregma; lateral, 0.8 mm; 4.0 mm ventral to dura). Anchor
screws and acrylic cement were used for fixation of the guide cannulas.
The body temperature of the animals was monitored by rectal probe and
maintained at 37° C. The rats were allowed to recover from surgery
for 2 days, housed singly in cages. On the day of the experiment a
microdialysis probe (CMA/12, 0.5 mm diameter, 3 mm length) was inserted
through the guide cannula.

[0164] The probes were connected via a dual channel swivel to a
microinjection pump. Perfusion of the microdialysis probe with filtered
Ringer solution (145 mm NaCl, 3 mM KCl, 1 mM MgCl2, 1.2 mM
CaCl2 containing 0.5 μM neostigmine) was begun shortly before
insertion of the probe into the brain and continued for the duration of
the experiment at a constant flow rate of 1 μl/min. After 180 min of
stabilisation, the experiments were initiated. Dialysates were collected
every 20 min. After the experiments the animals were sacrificed, their
brains removed, frozen and sliced for probe placement verification.

[0168] In single injection experiments the mean value of 3 consecutive ACh
samples immediately preceding compound administration served as the basal
level for each experiment and data were converted to percentage of basal
(mean basal pre-injection values normalized to 100%). The data are
presented in FIGS. 25a and 25b.

[0169] The data presented in FIG. 24 show unexpected drops in the
acetycholine levels (see e.g. 8 mg/kg) which are difficult to explain and
which are ascribed to experimental uncertainty. Overall, both data sets
from example 2F and 2G show the same, i.e. a dose dependent increase in
the extra-cellular acetylcholine levels in the brain. This pre-clinical
finding is expected to translate into an improvement in cognition in a
clinical setting useful e.g. in the treatment of diseases characterised
by a cognitive impairment, such as e g Alzheimer's patients, partial
responders, cognitive impairment etc.

Example 3

Effect on Neuropatic Pain

[0170] To demonstrate an efficacy against neuropathic pain, the compound
of the present invention was tested in the formalin model of neuropathic
pain [Neuropharm., 48, 252-263, 2005; Pain, 51, 5-17, 1992]. In this
model, mice receive an injection of formalin (4.5%, 20 μl) into the
plantar surface of the left hind paw and afterwards are placed into
individual glass beakers (2 l capacity) for observation. The irritation
caused by the formalin injection elicits a characteristic biphasic
behavioural response, as quantified by the amount of time spent licking
the injured paw. The first phase (˜0-10 minutes) represents direct
chemical irritation and nociception, whereas the second (˜20-30
minutes) is thought to represent pain of neuropathic origin. The two
phases are separated by a quiescent period in which behaviour returns to
normal. Measuring the amount of time spent licking the injured paw in the
two phases assesses the effectiveness of test compounds to reduce the
painful stimuli.

[0171] Eight C57/B6 mice (ca. 25g) were tested per group. Table 4 below
show the amount of time spent licking the injured paw in the two phases,
i.e. 0-5 minutes and 20-30 minutes post formalin injection. The amount of
compound administered is calculated as the free base.

[0172] The data in table 4 shows that the compound of the present
invention has little effect in the first phase representing direct
chemical irritation and nociception. More notably, the data also show a
clear and dose dependent decrease in the time spent licking paws in the
second phase indicating an effect of the compound of the present
invention in the treatment of neuropathic pain.

Example 4

Capsules

[0173] 4-[2-(4-methylphenylsulfanyl)-phenyl]piperidine hydrobromide was
mixed with microcrystalline cellulose in a first step. In a second step
magnesium stearate was mixed in. Capsules with four strengths were
prepared--the active ingredient is stated as the free base